Growth In Liquid Medium Research Articles

Lignocellulose-Adapted Endo-Cellulase Producing Streptomyces Strains for Bioconversion of Cellulose-Based Materials.

Twenty-four Actinobacteria strains, isolated from Arundo donax, Eucalyptus camaldulensis and Populus nigra biomass during natural biodegradation and with potential enzymatic activities specific for the degradation of lignocellulosic materials, were identified by a polyphasic approach. All strains belonged to the genus Streptomyces (S.) and in particular, the most highly represented species was Streptomyces argenteolus representing 50% of strains, while 8 strains were identified as Streptomyces flavogriseus (synonym S. flavovirens) and Streptomyces fimicarius (synonyms Streptomyces acrimycini, Streptomyces baarnensis, Streptomyces caviscabies, and Streptomyces flavofuscus), and the other four strains belonged to the species Streptomyces drozdowiczii, Streptomyces rubrogriseus, Streptomyces albolongus, and Streptomyces ambofaciens. Moreover, all Streptomyces strains, tested for endo and exo-cellulase, cellobiase, xylanase, pectinase, ligninase, peroxidase, and laccase activities using qualitative and semi-quantitative methods on solid growth medium, exhibited multiple enzymatic activities (from three to six). The 24 strains were further screened for endo-cellulase activity in liquid growth medium and the four best endo-cellulase producers (S. argenteolus AE58P, S. argenteolus AE710A, S. argenteolus AE82P, and S. argenteolus AP51A) were subjected to partial characterization and their enzymatic crude extracts adopted to perform saccharification experiments on A. donax pretreated biomass. The degree of cellulose and xylan hydrolysis was evaluated by determining the kinetics of glucose and xylose release during 72 h incubation at 50°C from the pretreated biomass in the presence of cellulose degrading enzymes (cellulase and β-glucosidase) and xylan related activities (xylanase and β-xylosidase). The experiments were carried out utilizing the endo-cellulase activities from the selected S. argenteolus strains supplemented with commercial β-gucosidase and xylanase preparations from Genencore (Accellerase BG and Accellerase XY). Cellulose and xylan conversion, when conducted using commercial (hemi)cellulases, gave glucose and xylose yields of 30.17 and 68.9%, respectively. The replacement of the cellulolytic preparation from Genencor (Accellerase 1500), with the endo-cellulase from S. argenteolus AE58P resulted in almost 76% of the glucose yield obtained in the presence of the commercial counterpart. Due to the promising results obtained by using the enzymatic crude extracts from S. argenteolus AE58P in the pretreated A. donax saccharification experiments, the proteins putatively responsible for endo-cellulase activity in this strain were identified by proteomics. Several proteins were confidently identified in different Streptomyces spp., eight of which belong to the class of Carbohydrate active enzymes. Overall results highlighted the biotechnological potential of S. argenteolus AE58P being an interesting candidate biocatalyst-producing bacterium for lignocellulose conversion and production of biochemicals and bioenergy.

Vision Marker-Based In Situ Examination of Bacterial Growth in Liquid Culture Media.

The detection of bacterial growth in liquid media is an essential process in determining antibiotic susceptibility or the level of bacterial presence for clinical or research purposes. We have developed a system, which enables simplified and automated detection using a camera and a striped pattern marker. The quantification of bacterial growth is possible as the bacterial growth in the culturing vessel blurs the marker image, which is placed on the back of the vessel, and the blurring results in a decrease in the high-frequency spectrum region of the marker image. The experiment results show that the FFT (fast Fourier transform)-based growth detection method is robust to the variations in the type of bacterial carrier and vessels ranging from the culture tubes to the microfluidic devices. Moreover, the automated incubator and image acquisition system are developed to be used as a comprehensive in situ detection system. We expect that this result can be applied in the automation of biological experiments, such as the Antibiotics Susceptibility Test or toxicity measurement. Furthermore, the simple framework of the proposed growth measurement method may be further utilized as an effective and convenient method for building point-of-care devices for developing countries.

Carbon Sequestration in Response to Grassland–Shrubland–Turfgrass Conversions and a Test for Carbonate Biomineralization in Desert Soils, New Mexico, USA

Core Ideas Soil carbonate is typically viewed as a soil mineral that requires centuries to accumulate; our study indicates that it can be generated in months to years. If the source of Ca is directly from silicate minerals, soil carbonate could be managed to sequester atmospheric CO2. Carbon‐13 values can change in less than a decade for both soil organic and inorganic C. This study uses an experimental pedology approach using controls, treatments, and replications. This study uses an experimental pedology approach to examine (i) how the conversion of native C4 grassland to C3 woody shrubs then to irrigated C4 turfgrass affects both soil organic C (SOC) and soil inorganic C (SIC) and (ii) whether SIC can be enhanced by microbial biomineralization. Three sites were studied in the Chihuahuan Desert of New Mexico. At each site, SOC, SIC, and their δ13C values were measured on control soil samples and compared to samples treated with liquid growth medium and Ca(OH)2 at 10 and 40 cm. The treated samples were left to react for 1 mo in February, May, and August of 2014. Using a space‐for‐time substitution, soil organic C decreased when native grasslands converted to desert shrubs, then sharply increased after desert shrubs were converted to irrigated turfgrass. Most surprising, however, was the increase of SIC in the turfgrass site, which doubled in 6 yr. The δ13C values of both SOC and SIC reflected the change from C4 to C3 then back to C4 vegetation and showed how rapidly SOC and SIC can change their isotopic signatures. Soil inorganic C formation was slightly higher for the liquid growth medium, but no statistically significant differences were observed between the treatments and control samples. In addition, no biomineralization was observed with microscopy, perhaps because the 1‐mo reaction time was too short and the amount applied was too small. Although SIC is typically viewed as a soil mineral that requires centuries to accumulate, our study indicate that SIC can be generated in months to years if the soil environment is suitable.

Stiffness of a biocemented sand at small strains

This paper presents the triaxial testing results of a study in which biocementation processes were employed to observe the behaviour at small strains of sand matrix. Biocementation was achieved using the microorganism Sporosarcina pasteurii, an aerobic bacteria growth in natural soil deposits. The microbes were introduced to the sand specimens in a liquid growth medium with three different amounts of urea and dissolved calcium sources. Subsequent cementation treatments were passed through the specimen under confining pressure. The results were assessed by local strain measurements using LVDTs. A series of undrained compression triaxial tests indicate that at small strain levels the observed stiffness-strain behaviour of biocemented sand specimens appeared to be significantly high for all three treatment levels. The response of biocemented sands was found to be similar to that of calcite and lime cemented specimens, which represent conventional cementing agents. Scanning electron microscopy pictures and energy dispersive X-ray were taken to verify the formation of cement at three different concentrations.

Enhanced plasmid loss in bacterial populations exposed to the antimicrobial compound irgasan delivered from interpenetrating polymer network silicone hydrogels

The spread of antimicrobial resistance, usually mediated by horizontal transfer of plasmids, limits the options of treating bacterial infections and thereby poses a crucial human health problem. The disturbance of plasmid stability within bacterial species in clinical environments serves as a novel strategy to reduce the development and dissemination of antibiotic resistance.We tested the ability of irgasan to destabilize plasmids from Escherichia coli K-12 cells when added directly into liquid growth medium at concentrations below levels of marked bacterial growth inhibition, or when released into liquid growth medium from irgasan-impregnated Interpenetrating Polymer Network (IPN) silicone hydrogel objects, a novel technology developed as drug-delivery platform. IPN-mediated irgasan-release was indirectly monitored as the extent of plasmid loss from bacterial cells during a 24-hour period or during repeated exposure to new irgasan-loaded IPN devices every 24h for a total of 10days. The cells were genetically modified so that plasmid loss could be quantified by applying a combination of fluorescence-based reporter gene technology and flow cytometry.When exposing bacterial cells to the irgasan-impregnated IPNs for 24h, we observed a modest (2.8–4.7%), but significant (P<0.05), plasmid loss as well as an inhibition of bacterial growth, both gradually increasing with increasing impregnation concentration. Repeated exposure to irgasan-impregnated IPNs drastically increased the plasmid loss of up to 83%, but cells adapted over time, which indicated the limitations of this specific drug for future medical applications. This study, however, illustrates the ability of IPNs to release an impregnated compound into a liquid suspension to induce a significant biological impact on growing bacterial cells.

English

Low Phosphorus availability in soil is worldwide a frequent major constraint for crops. Rhizobial strains, beneficial N2-fixing symbiotic partners of legumes, were reported to solubilize both organic and inorganic complex phosphates. Thus they may play a significant role as plant growth-promoting rhizobacteria (PGPR) in the biofertilization of crops. Natural rock phosphate may play a key role in sustainable agriculture, resulting in economic and environmental benefits. This work aims to evidence the capacity of rhizobial strains nodulating Vicia faba L. plants in Marrakech-Haouz region (Morocco) to solubilize Moroccan rock phosphate in vitro in either agar or liquid medium and to evaluate and compare the effect of these strains on growth and phosphorus uptake by V. faba plants of two Moroccan varieties (Defes and Aguadulce). Four rhizobial strains, contrasting for phosphate solubilization on agar medium, were further studied on liquid medium. RHOF147 and RHOF174 strains formed clear halos around colonies (1.37 cm and 1.2 respectively after 15 days); opposite RHOF170 and RHOF171 strains did not produce halo. The four strains were able to mobilize rock phosphate for growth in liquid medium (up to 0.59 mg/L of assimilated phosphorus). Lower pH values and most mobilized phosphorus production were registered for RHOF147 and RHOF174 strains. By screening the four rhizobial strains for growth on Chrome Azurol S (CAS) medium, only RHOF171 strain formed orange halo indicating its capacity to produce siderophores. Greenhouse experiments were undertaken including comparison of inoculated plants with these four rhizobial strains in the presence of rock phosphate as sole P source versus non-inoculated plants growing on KH2PO4 as sole P source (P-fertilized plants). Results show that the root length, the root dry weight and the ratio root /shoot dry weights were generally higher in the inoculated plants than in the P-fertilized plants, indicating the adaptation of the inoculated plants to phosphorus deficiency conditions. The effect of rock phosphate-solubilizing bacteria on plant growth depends on the symbiotic combination of the inoculated strain and the plant genotype. As for phosphorus uptake, rhizobial strains made phosphorus available to the plant at significant increased concentrations in some symbiotic combinations (e.g. Aguadulce-RHOF147). The use of selected symbiotic combinations having the capacity to solubilize rock phosphate may improve soil fertility and phosphorus availability to plants. (Resume d'auteur)

English

Phosphorus solubilizers naturally acidify rhizospheric soil and increase phosphorus availability; therefore, their evaluation may help to reduce phosphorus fertilizer use. This work aimed to evaluate the different selection methods and select inorganic phosphorus-solubilizing bacteria as potential plant-growth promoters. Bacterial isolates obtained from sugarcane roots and soil were tested using solid growth media containing bicalcium phosphate and Irec&ecirc; Apatite ground rock phosphate as phosphorus sources. Seven isolates with high (3), moderate (3) and low solubilization indices (1) and the Pseudomonas fluorescens R-243 strain were tested in two liquid growth media, followed by the pH and soluble P in the solution. The same isolates, in the absence of inoculation, were tested in Leonard jars with two high- and low-solubility sources using cowpea as a test species. Forty-four days after planting aboveground dry mass, the phosphorus content and total aboveground phosphorus and substratum phosphorus contents were evaluated. The growth media affected phosphorus solubilization by the bacteria. Evaluation of liquid media was the most reliable method for analyzing bicalcic phosphorus solubilization by the bacteria not linked to pH reduction. Isolates UAGC 17, 19 and 65 should be better studied because they were the best solubilizers in culture media; however, they did not demonstrate the same efficiency when inoculated on cowpea. Key words: Solubilization, phosphate, P-solubilizing-microorganisms.

Increasing phosphorus concentration in the extraradical hyphae of Rhizophagus irregularis DAOM 197198 leads to a concomitant increase in metal minerals.

Plants associated with arbuscular mycorrhizal fungi (AMF) acquire phosphorus via roots and extraradical hyphae. How soil P level affects P accumulation within hyphae and how P in hyphae influences the accumulation of metal minerals remains little explored. A bi-compartmented in vitro cultivation system separating a root compartment (RC), containing a Ri T-DNA transformed carrot root associated to the AMF Rhizophagus irregularis DAOM 197198, from a hyphal compartment (HC), containing only the extraradical hyphae, was used. The HC contained a liquid growth medium (i.e., the modified Strullu-Romand medium containing P in the form of KH2PO4) without (0μM) or adjusted to 35, 100, and 700μM of KH2PO4. The accumulation of P and metal minerals (Ca, Mg, K, Na, Fe, Cu, Mn) within extraradical hyphae and AMF-colonized roots, and the expression of the phosphate transporter gene GintPT were assessed. The expression of GintPT in the extraradical hyphae did not differ in absence of KH2PO4 or in presence of 35 and 100μM KH2PO4 in the HC but was markedly reduced in presence of 700μM KH2PO4. Hyphal P concentration was significantly lowest in absence of KH2PO4, intermediate at 35 and 100μM KH2PO4 and significantly highest in presence of 700μM KH2PO4 in the HC. The concentrations of K, Mg, and Na were positively associated with the concentration of P in the extraradical hyphae developing in the HC. Similarly, P concentration in extraradical hyphae in the HC was related to P concentration in the growth medium and influenced the concentration of K, Mg, and Na. The accumulation of the metal mineral K, Mg, and Na in the extraradical hyphae developing in the HC was possibly related to their function in neutralizing the negative charges of PolyP accumulated in the hyphae.

A high throughput screen of a natural product library for the discovery of novel antibiotics that target Enterococcus faecalis.

Antibiotic resistance is a natural phenomenon that serves as a threat to public health. The investigation and discovery of novel antibiotic compounds is essential for battling the trend of decreasing antibiotic efficacy. The National Center for Natural Products Research (NCNPR) has assembled a library of organic compounds derived from plants, fungi, marine, and algae species with potential antibacterial properties. In partnership with NCNPR, we have developed a rapid and effective method for screening large quantities of organic compounds for antibacterial efficacy against Enterococcus faecalis. E. faecalis is a gram positive opportunistic pathogen responsible for various hospital acquired infections. To identify extracts that were inhibitory against E. faecalis, bacteria were inoculated into 96-well plates containing liquid growth medium. A small volume of each compound was added to a corresponding well in the 96-well plate. After 24 hour incubation at 37o C, bacteria growth was measured using a plate reader.Wells lacking treatment or containing antibiotics recognized as inhibitors of E. faecalis were used to identify normal or inhibited growth levels correspondingly. Compounds are currently being evaluated for their ability to inhibit bacterial growth. Further investigations will involve the characterization and a detailed analysis of the antimicrobial efficacy of these compounds.

A high throughput screen of a natural product library for the discovery of novel antibiotics with efficiacy against Klebsiella pneumoniae.

Klebsiella pneumoniae is a gram negative bacterium in the enterobacteriaceae family. This organism is an opportunistic pathogen that has been associated with antibiotic resistance in recent years. The discovery of new antibiotics is critical to combat this rising trend in resistance. The National Center for Natural Products Research (NCNPR) has assembled a library of over 2500 extracts derived from plants, fungi, marine, and algae species with potential antibacterial properties. In partnership with NCNPR, we have developed a rapid and effective method for screening large quantities of organic compounds for antibacterial sensitivity against K. pneumoniae. Bacteria were inoculated into a microtiter plate containing liquid growth medium. A small volume of each compound was added to a corresponding well in the microtiter dish.After incubation at 37°C, bacteria growth was measured using a plate reader. Wells lacking treatment or containing antibiotics known to be effective at inhibiting K. pneumoniae were used to identify normal or inhibited growth levels respectively. At least four extracts exhibited potential for inhibiting K. pneumoniae: blanket flower, smallflower globemallow, Arizona walnut, and tree fern. Future investigations will involve the characterization and a detailed analysis of the antimicrobial efficacy of these compounds.

Identification and functional analysis of endogenous nitric oxide in a filamentous fungus

In spite of its prevalence in animals and plants, endogenous nitric oxide (NO) has been rarely reported in fungi. We present here our observations on production of intracellular NO and its possible roles during development of Neurospora crassa, a model filamentous fungus. Intracellular NO was detected in hypha 8–16 hours after incubation in Vogel’s minimal liquid media and conidiophores during conidiation using a fluorescent indicator (DAF-FM diacetate). Treatment with cPTIO, an NO scavenger, significantly reduced fluorescence levels and hindered hyphal growth in liquid media and conidiation, whereas exogenous NO enhanced hyphal extension on VM agar media and conidia formation. NO scavenging also dramatically diminished transcription of con-10 and con-13, genes preferentially expressed during conidiation. Our results suggest that intracellular NO is generated in young hypha growing in submerged culture and during conidia development and regulate mycelial development and conidia formation.

Synthesis, characterization and evaluation of antibacterial efficacy of zinc oxide nanoparticles

Objective: Objective of the study was to synthesize and characterize Zinc oxide (ZnO) nanoparticles (NP), and to evaluate their application on some bacterial strains. Methods: ZnO NP was synthesized by chemical methods. Then decomposed by using conventional heating process. The detailed characterization of the nanoparticles was performed using FT-IR, UV-Vis spectroscopy, X-Ray Diffraction analysis and XRF. From the analysis of XRD pattern, UV-VIS spectroscopy and XRF, the formation of nanoparticles was confirmed. Antibacterial assay of synthesized ZnO NP was carried out both in liquid and solid growth medium against a gram positive ( Staphylococcus aureus ) and a gram negative ( Escherichia coli ) bacteria using disc diffusion assay method. Effect of antibacterial activity was observed by zone of inhibition around the antibiotic discs of ZnO NP. Results: ZnO NP was characterized by the different spectral analysis of the synthesized product. ZnO NPs reveal good antibacterial activity against S. aureus and E. coli . Kinetic studies were conducted on growth bacteria by loading ZnO NP to S. aureus and E. coli with this concentration to study the kinetic of growth behavior which showed that NP produced toxicity on both bacteria and therefore the growth was inhibited. Conclusions: The inhibition of growth of the organisms by ZnO nanoparticles suggests that it could potentially be used as an effective antibacterial agent and as well can be used in the protection of agricultural and food safety. Future studies may be aimed at the further evaluation to establish the nanoparticles as potential antimicrobial agent.

SU‐F‐SPS‐08: Measuring the Interaction Of DDR Cell Receptors and Extracellular Matrix Collagen in Prostate Cells

Purpose:Discoidin domain receptors (DDR) have recently been recognized as important players in cancer progression. DDRs are cell receptors that interact with collagen, an extracellular matrix (ECM) protein. However the detailed mechanism of their interaction is unclear. Here we attempted to examine their interaction in terms of structural (surface topography), mechanical (rupture force), and kinetic (binding probability) information on the single molecular scale with the use of atomic force microscopy (AFM).Methods:The Quantitative Nano‐mechanical property Mapping (QNM) mode of AFM allowed to assess the cells in liquid growth media at their optimal physiological while being viable. Human benign prostate hyperplasia (BPH‐1) cell line was genetically regulated to suppress DDR expression (DDR‐ cells) and was compared with naturally DDR expressing cells (DDR+).Results:Binding force measurements (n = 1000) were obtained before and after the two groups were treated with fibronectin (FN), an integrin‐inhibiting antibody to block the binding of integrin. The quantification indicates that cells containing DDR bind with collagen at a most probable force of 80.3–83.0 ±7.6 pN. The probability of them binding is 0.167 when other interactions (mainly due to integrin‐collagen binding) are minimized.Conclusion:Together with further force measurements at different pulling speeds will determine dissociation rate, binding distance and activation barrier. These parameters in benign cells provides some groundwork in understanding DDR's behavior in various cell microenvironments such as in malignant tumor cells.Funding supported by Richard Barber Interdisciplinary Research Program of Wayne State University

Antimicrobial Activity for Excretion and Secretion of the Greenbottle Fly Larvae Lucilia Sericata ( Meigen ) ( Diptera : Calliphoridae )

Sterile larval excretion/secretion (ES) exhibited antibacterial activity against some species of bacteria. They were shown to inhibit the growth of Gram-positive bacteria Staphylococcus aureus and Bacillus subtilis Gram-negative bacteria Pseudomonas aeruginosa, Escherichia coli and Klebsiella pneumoniae and Fungi Geotricum candidum and Aspergillus fumigatus thus exhibited limited inhibitory effect towards Gram-positive bacteria Streptococcus pyogenes and Staphylococcus epidermidis and Gram-negative Proteous vulgaris and Fungi Syncephalastrum racemosum, Candida albicans, that effect was slowed down when challenged with secretion on a solid media but no zone of complete inhibition was detectqd. Growth inhibiting activity was determined in liquid growth media using the Gram-positive, Gram-negative bacterial and fungal strains as indicator organisms.

Split-agar assay of antifungal soil microbial metabolites

Abstract Soil microorganisms suppress soil borne plant pathogenic fungi through various mechanisms. There is no appropriate, integrated method to easily quantify soil health in terms of disease control ability. A novel assay to quantify the ability of soil to inhibit fungal pathogens is described. The technique is easy to use routinely in soil biology investigations for soil quality testing as it offers an integrated expression of suppressiveness as actidione equivalents per gram of soil. Soil samples were inoculated into liquid growth medium and incubated; supernatants were filter sterilized and assayed in split agar against Macrophomina phaseolina to record colony radius. The antifungal activity of the soils varied widely ranging from 0.02 to 2.80 mg actidione equivalents g −1 soil, of which 81–98% was heat labile. The test will be a useful aid in decision support for reducing the use of chemical control agents and promote sustainable farming practices.

Effects of Lipopolysaccharide Core Sugar Deficiency on Colanic Acid Biosynthesis in Escherichia coli.

When 10 Escherichia coli mutant strains with defects in lipopolysaccharide (LPS) core biosynthesis were grown on agar medium at 30°C, four of them, the ΔwaaF, ΔwaaG, ΔwaaP, and ΔwaaB strains, formed mucoid colonies, while the other six, the ΔwaaU, ΔwaaR, ΔwaaO, ΔwaaC, ΔwaaQ, and ΔwaaY strains, did not. Using light microscopy with tannin mordant staining, the presence of exopolysaccharide around the cells of the mutants that formed mucoid colonies could be discerned. The ΔwaaF mutant produced the largest amounts of exopolysaccharide, regardless of whether it was grown on agar or in liquid medium. The exopolysaccharide was isolated from the liquid growth medium of ΔwaaF cells, hydrolyzed, and analyzed by high-performance liquid chromatography with an ion-exchange column, and the results indicated that the exopolysaccharide was consistent with colanic acid. When the key genes related to the biosynthesis of colanic acid, i.e., wza, wzb, wzc, and wcaA, were deleted in the ΔwaaF background, the exopolysaccharide could not be produced any more, further confirming that it was colanic acid. Colanic acid could not be produced in strains in which rcsA, rcsB, rcsD, or rcsF was deleted in the ΔwaaF background, but a reduced level of colanic acid production was detected when the rcsC gene was deleted, suggesting that a change of lipopolysaccharide structure in ΔwaaF cells might be sensed by the RcsCDB phosphorelay system, leading to the production of colanic acid. The results demonstrate that E. coli cells can activate colanic acid production through the RcsCDB phosphorelay system in response to a structural deficiency of lipopolysaccharide. Lipopolysaccharide and colanic acid are important forms of exopolysaccharide for Escherichia coli cells. Their metabolism and biological significance have been investigated, but their interrelation with the cell stress response process is not understood. This study demonstrates, for the first time, that E. coli cells can activate colanic acid production through the RcsCDB phosphorelay system in response to a structural change of lipopolysaccharide, suggesting that bacterial cells can monitor the outer membrane integrity, which is essential for cell survival and damage repair.

Optical fiber Fabry–Perot interferometer for microorganism growth detection

Abstract An optical fiber Fabry–Perot interferometer (FPI) based on hollow-core photonic crystal fiber (HCPCF) for microorganism growth detection is proposed and demonstrated. The FPI is formed by splicing both ends of a short section of HCPCF to SMFs and cleaving the SMF pigtail to a proper length. By measuring the fringe contrast of interference pattern, the refractive index (RI) changes of analyte during microorganism growth can be obtained. RI response of the sensor was investigated theoretically and experimentally. It shows linear response with sensitivity of −136 dB/RIU and good repeatability. Temperature response was also tested and the result confirms the low temperature cross-sensitivity of the sensor. Detection of yeast growth in liquid medium by the FPI sensor was conducted and the result shows the characteristic of typical yeast growth curve. With its advantages of high RI sensitivity, low temperature cross-sensitivity, capability for real-time measurement and so on, this FPI sensor has great potential in biosensing.

In vitro evaluation of a novel bacteriophage cocktail as a preventative for bovine coliform mastitis

The objective of this study was to investigate the potential use of bacteriophage in preventing Escherichia coli mastitis on dairies. A cocktail consisting of 4 distinct bacteriophages was generated by screening against 36 E. coli isolates from dairy cows in Washington State with clinical mastitis. The bacteriophage significantly inhibited growth of 58% of the Washington State isolates and 54% of E. coli mastitis isolates from New York State, suggesting that the cocktail of phages had a relatively broad spectrum of action against relevant strains from 2 distinct geographies. The ability to suppress bacterial growth of these isolates in a liquid growth medium was not affected by the ratio of bacteriophage particles to bacterial cells (multiplicity of infection, MOI). For those E. coli that were completely inhibited by the phage cocktail, an MOI as low as 10had the same effect as 10µg/mL of ceftiofur on the growth rate of E. coli over a 12-h period using optical density measurements. A 3.3- to 5.6-log reduction of growth was achieved when E. coli was co-incubated with our phage cocktail in raw milk over a 12-h period at physiologic temperature. A modified gentamicin protection assay using bovine mammary epithelial cells provided a model to test whether bacteriophage could prevent cell attachment and invasion by chronic coliform mastitis strains. Pretreatment of cell cultures with the phage cocktail significantly reduced adhesion and intracellular survival of E. coli compared with controls. When combined with a bismuth-based teat sealant, the phage cocktail was able to inhibit bacterial growth when challenged with 1.6×103 cfu/mL of a clinical mastitis E. coli strain. In vitro results show bactericidal activity by our phage in raw milk and mammary tissue culture systems. Before a bacteriophage-based dry-cow treatment becomes a potential option for dairies, in vivo studies must be able to demonstrate that a specific dose of bacteriophage can protect cows from experimentally induced E. coli mastitis without inducing an inflammatory reaction.

Electron microscopy reveals a soluble hybrid network of individual nanocrystals self-anchored by bifunctional thiol fluorescent bridges.

Today, nanochemistry research of hybrid materials growth in liquid media represents a new challenge for tailoring specific nano-sized materials directly related to the hybrid electron-optical properties. Distinctive assumptions about the origin, the growth, and the functionalization of hybrid nanoparticles have recently been proposed by scientific research to attend the different aspects of observable behaviors. Therefore, appropriate morpho-structural observation of the hybrid nanoparticles is the most important factor for controlling the chemical and physical properties. Here, we report how the gold nanocrystals (Au-NCs) structurally covered by an outer layer material of 9,9-didodecyl-2,7-bisthiofluorene (FL) bifunctional stabilizer evolve into a self-organized 2D-network as a function of different nano-structural features. Detailed morpho-structural investigation of this hybrid material through electron microscopy techniques has been performed from the atomic-scale to hundreds of nanometers. The experimental information gathered allowed us to figure out the evolution growth of the gold-FL nanoparticles (AuFL-NPs) from the early stage of the gold-organic nucleation to the final assembled bi-dimensional network. The reported results represent a valuable background toward the full comprehension of growth mechanisms of organic-inorganic materials responsible for the final chemical and physical properties.

Effect of co-culture with enterocinogenic E. faecium on L. monocytogenes key virulence gene expression

The aim of the present study was to assess the expression of key virulence genes during co-culture of L. monocytogenes with a bacteriocinogenic E. faecium strain in liquid growth medium. For that purpose, BHI broth was inoculated with 7 log CFU·mL –1 L. monocytogenes and 4, 5 or 6 log CFU·mL –1 E. faecium . Sampling took place after 8 and 24 h of incubation, corresponding to the maximum and minimum of enterocin production, respectively. The RNA was extracted, stabilized and expression of prfA , sigB , hly , plcA , plcB , inlA , inlB , inlC and inlJ , was assessed by RT-qPCR. Most of the genes were downregulated during co-culture at 5 °C. Moreover, a statistically significant effect of the inoculum level was evident in most of the cases. On the contrary, no effect on the transcription level of most of the genes was observed during co-culture at 37 °C.